PROJECT SUMMARY The sarcomeric myosins responsible for muscle contraction are among the most well studied proteins, yet we are still discovering new roles for this class of molecule. Myosin heavy chain 7b (MYH7b) is a recently identified gene belonging to the sarcomeric myosin family that appears to have evolved new roles in mammalian non-muscle tissue. In mammals, MYH7b protein is only expressed in specialized tissues such as extraocular muscle and muscle spindles and, surprisingly, is found in the inner ear and brain. These observations are highly unusual as no other muscle myosin has ever been found in non-sarcomeric tissues. Further, mutations in MYH7b cause hearing loss in humans with no apparent muscle phenotypes, indicating that MYH7b has an important yet undefined role in non-muscle tissue. Intriguingly, in snakes, MYH7b is found in the sarcomeres of cardiac and skeletal muscles. Despite these divergent roles in reptiles and mammals, MYH7b shares high sequence identity between these classes of vertebrates, so it is unclear how mammalian MYH7b function may deviate in non-muscle tissues from its expected sarcomeric role. The goal of this proposal is to identify the role and activity of MYH7b in mammalian non-muscle tissue and understand how mutations in MYH7b contribute to hearing loss. I hypothesize that distinct biophysical and biochemical properties allow mammalian MYH7b to function in non-muscle environments, and that mutations in MYH7b disrupt hearing by causing this protein to interfere with normal auditory processes. In Aim 1, I will determine the physiological role of MYH7b in mammals using mouse models. First, I will investigate the role of MYH7b in wild type mice by determining subcellular localization, protein interactions, and structures formed by MYH7b in the inner ear. Next, I will investigate any anatomical and behavioral defects in MYH7b null mice that our lab is currently maintaining. Finally, I will use CRISPR/Cas gene editing to introduce the human MYH7b mutations that cause hearing loss into mice and assess hearing function and any resultant anatomical or behavioral defects. I anticipate this work will reveal how MYH7b functions in the auditory system, which will provide a foundation for understanding MYH7b function in the mammalian brain. In Aim 2, I will investigate the molecular function of MYH7b by comparing the biophysical and biochemical activities of recombinant python and human MYH7b protein. These analyses will address the question of whether these proteins have similar molecular functions, or whether the mammalian MYH7b activity has diverged from other sarcomeric myosins. I will also determine the functional impact of the two hearing loss mutations (one in the catalytic motor domain and one in the structural rod domain of the myosin) on motor properties and the molecule's ability to self assemble into ordered structures. Collectively, this work will provide a comprehensive understanding of the role of mammalian MYH7b and its activity in non-muscle tissue at the molecular, cellular, and whole organism level as well as provide a foundation for a better understanding of MYH7b evolution and divergence across species.